Semiconductor manufacturing and other "clean" industries require extremely pure water such as ultra-pure water or deionized water. Numerous methods of obtaining ultra-pure water and deionized water are known in the art. For example, ultra-pure water may be obtained by a physical filtration utilizing a filter having filtering apertures of a designated level. In the preparation of deionized (DI) water, ions contained in water are separated or adsorbed either through electrochemical methods or with ion resin layers.
In addition to particulate and ionic contaminants, water used in industries such as semiconductor manufacturing is also threatened by microbial contamination. While the presence of microorganisms, per se, in otherwise clean water is not desirable, their presence is also not necessarily disastrous, in that the water is usually in contact with the semiconductor component being manufactured for such a brief amount of time that damage to the component is negligible. Far more dangerous to clean industrial processes is the presence of biofilm that may be produced by microorganisms present in, for example, ultrapure or deionized water, ultrapure water preparing apparatuses, ultrapure water piping systems, production lines or semiconductor materials in the manufacturing process. Biofilm is a gel state layer containing microorganisms growing on the boundary between liquid and solid phases in an aqueous environment (e.g., the surface of a substratum in a water piping system), and attached to the surface by aggregates of extracellular polymer substances (also referred to as exopolymer polysaccharides, or EPS) produced by the organisms. Notably, biofilm may contain a large number of microorganisms (about 10.sup.7 to 10.sup.11 cells/ml of biofilm mass), even though few microorganisms (as few as 1 to 10 CFU/ml) are simultaneously growing in the bordering aqueous phase.
Generally, biofilm encloses the microorganisms and primarily consists of water (70 to 95% by weight of wet weight) and an organic substance such as a polysaccharide (70 to 95% by weight of dry weight). Biofilm may be formed uniformly on the entire surface of the substratum, or may be formed in patchy patterns in thicknesses of up to about several hundreds micrometers. Since biofilm prevents the diffusion of dissolved oxygen, aerobic microorganisms contained in biofilm as thin as about 50-150 .mu.m become anaerobic.
Unattached microorganisms present in purified water or water systems may be removed, to a certain extent, by sterilization processes known to those skilled in the art. Unfortunately, simple sterilization is not sufficient to remove attached microorganisms and the biofilm produced by such microorganisms from such environments. One reason that sterilization is ineffective in removing attached microorganisms is that physiologically inactive microorganisms are easily adsorbed onto surfaces present in aqueous environments, thus serving as additional surfaces that newly introduced microorganisms may adsorb to. Microorganisms on the surface of biofilm are difficult to kill or remove by sterilization because they form matrices with other microorganisms and the EPS produced thereby.
At present, deposits of attached microorganisms and biofilms produced thereby are removed by two primary methods: (1) chemical methods utilizing oxidizing agents, biodispersants, surfactant and enzymes to weaken the interaction between the surface of an object and the biofilm matrix; and (2) physical methods such as shear force, mechanical methods and applications of ultrasonic energy that remove microorganisms and biofilms from fouled surfaces, with chemical methods being used primarily in the semiconductor manufacturing industry. In order to be effective, biocidal agents used in chemical methods must eliminate microorganisms efficiently before a rapid multiplication of the microorganisms can occur. The agents used in such methods are preferably safe and easily handled by operators, and mild enough to prevent physical and/or chemical damage to the system being sterilized/decontaminated. Furthermore, the biocidal agents must be easily removable from the systems being sterilized, in that the agents themselves may be considered contaminants if not removed just after the sterilization.
In current practice, semiconductor manufacturing companies using chemical methods for removing biofilms from their ultrapure water supply lines usually utilize hydrogen peroxide as the biocidal agent of choice. Hydrogen peroxide decomposes into water and oxygen, and thus does not remain after the sterilization, causing little or no corrosion in the pipe lines. Although the decontamination of water supply lines would be most effective if high concentrations of hydrogen peroxide were applied to the pipelines under high temperature conditions, hydrogen peroxide is generally used at relatively low concentrations (about 1%), so as not to produce harmful gases caused by the reaction of hydrogen peroxide and organic materials. Furthermore, temperatures of about 30.degree. C. are generally used in the hydrogen peroxide decontamination procedures because higher temperatures may cause either damage to the pipe lines themselves, or the discharge of harmful organic or inorganic materials from drainage pipes.
While offering certain advantages, the use of hydrogen peroxide as a chemical decontaminant to remove biofilm may also be problematic. Highly concentrated hydrogen peroxide is expensive, and its removal after decontamination procedures is time-consuming, often involving a complete shut-down of the manufacturing line. Moreover, there are currently no reliable standards for the use of hydrogen peroxide (e.g., standards of effective concentrations, duration and frequency of application, etc.) as a decontaminant for removing biofilm. Optical standards for sterilization/decontamination using hydrogen peroxide are difficult to determine on a systematic basis because different sterilization conditions may be required for individual production lines. When hydrogen peroxide is used to decontaminate a water supply line but it is later determined that the decontamination procedure has been ineffective, another, more toxic chemical agent such as formaldehyde must then be used to ensure sufficient decontamination. Such an additional step is costly and time consuming, but could be avoided if reliable standards for the of hydrogen peroxide could be established.
In order to monitor the efficacy of the sterilization process, numerous samples must be gathered from the ultrapure water pipe lines after each change in operating conditions in order to determine the number of surviving microorganisms (and thus the effectiveness of the decontamination procedure). After these samples are collected (e.g., by filtration, by collection from containment surfaces, or by other means known in the art), the microorganisms contained therein must then be cultured in order to accurately determine the concentration of microorganisms present in the water supply. Unfortunately, samples taken from ultrapure water systems are notoriously difficult to culture, and standard plate-count techniques applied to cultures grown from such samples may not give an accurate estimate of microbial concentration. Moreover, samples taken from nutrient-poor environments such as ultrapure water lines, if culturable at all, may take several days to weeks to grow into detectable colonies. This kind of delay is clearly not acceptable in industries such as semiconductor manufacturing, where continual and on-line control is necessary. Samples taken from water lines to monitor decontamination efficacy may be examined by microscopy in order to detect the presence of microorganisms. However, this process is not only time consuming, but also requires the presence of a concentration of microorganisms that may simply not be present in the sample, though the water line may actually be contaminated on its surfaces by biofilm.
It is apparent that standards for the effective use of hydrogen peroxide are desirable, as are methods and agents for the determination of the efficacy of a decontamination process using hydrogen peroxide. Such methods and standards could be established by using an indicator microorganism that exhibits a predictable resistance (i.e., a standardized survival curve) to hydrogen peroxide. Such an indicator microorganism would have to be an aerobic microorganism that is easily identified, analyzed, cultured and maintained. The microorganism would also have to be more strongly resistant to hydrogen peroxide than other microorganisms that are possible contaminants in, for example, an ultrapure water supply line. Finally, the indicator microorganism would be required to exhibit predictable, repeatable, and, preferably, linear survival curves against hydrogen peroxide.
Certain microorganisms such as Bacillus stearothermophilus NCIMB 8224 have been previously proposed as indicator microorganisms for monitoring the efficacy of hydrogen peroxide decontamination. However, a need exists for additional microorganisms that can be used for such purposes, and especially for microorganisms that exhibit linear survival curves in relation to varying concentrations of hydrogen peroxide.